Aspects of the reproductive biology and endocrinology of the substrate-spawning cichlid Tilapia zillii

This study investigated several, previously little-known, aspects of reproductive physiology and endocrinology in the substrate-spawning cÌchlid Tilapia zillii; a tilapia that is becoming increasingly popular in world aquaculture. Studies were undertaken in controlled laboratory aquaria, thereby reducing the potential influence of environmental variation evident in many previous field studies of this species. Analysis involved two strains of T. zillii: strain 'A' (T. zillii) and strain 'B' (formerly known as Tilapia tholloni). Spawning periodicity and total fecundity generally increased with fish size. Egg size varied within a narrow window and did not generally increase with fish size though fish weighing 100 - 200g tended to produce the largest eggs. The best estimate of spawning periodicity was considered to be 'mean days elapsed/spawn' as this figure was based upon both spawning and non-spawning fish in an experimental group. Mean days elapsed/spawn increased with increasing fish size and averaged 61.4 days and 37.5 days in strains 'A' and 'B' respectively. The shortest reproductive cycles observed were just 7 days and 6 days for strains 'A' and 'B' respectively. Total fecundity ranged from 461 - 11640 eggs/clutch. Mean total fecundity was 3606+/-280 in strain 'A' and 3560+/-243 in strain 'B'. Mean egg diameter was 1.5+/-0.04mm and 1.4+/-0.08mm in strains 'A' and 'B' respectively. Fecundity and egg size also varied over successive spawns in serial-spawning females but these variations did not appear to be related to spawning periodicity. Regression analysis revealed strong relationships between fish size (weight and length) and total fecundity, relative fecundity and total egg volume. Relationships between fish size and egg size were generally much weaker. Fecundity and egg size were related to the length of the preceding inter-spawn-interval (ISI) in fish of certain weight categories but not others, providing limited evidence that length of ISI may in par, control fecundity and egg size in this species. Ovarian recrudescence was classified into ten distinct developmental stages based upon oocyte size, biochemical properties and structure. This classification scheme was comparable to classification schemes developed for other teleosts but represents the first detailed description of oocyte growth in a substrate-spawning tilapia. Radioimmunoassay and stereological analysis provided valuable and novel data concerning the dynamics of ovarian development in this species. Levels of 17ßoestradiol (E2) and testosterone (T) peaked within 6 days of spawning, suggesting that vitellogenesis began as early as day 2 or 3 post-spawning. By day 8, ovaries were dominated by large late-vitellogenic/maturing oocytes (stages 6 & 7) occupying 60 - 70% of the ovary. Gonadosomatic index (GSI) reached maximal levels by day 14. Since the proportion of stage 6/7 oocytes exhibited little change from day 8 onwards, it is suggested that pre-vitellogenic oocytes are recruited into vitellogenic growth immediately after spawning and complete vitellogenesis as early as day 8 postspawning. Analysis of serial-spawning fish found that initial post-spawn E2 and T peaks (on days 2 - 6) were much lower after the second spawning. Sex steroid levels were also found to be suppressed in confined T. zillii (i.e. where stocking densities were > lOkg/m3). Confined females failed to spawn but displayed a marked tendency to do so after transfer to individual aquaria. Serum E2 and T were suppressed during confinement but increased rapidly following transfer to individual aquaria (coincident with resumed spawning activity). It is suggested that levels of E2 and T under confinement are not sufficient to allow completion of vitellogenic growth and are most probably suppressed via a pheromonal mechanism. Finally, the present study investigated the effect of prolonged food restriction on various aspects of reproduction. T. zillii were rationed from first feeding and throughout the following 17 months. Despite very large differences in fish size, no significant differences were detected in total fecundity, egg diameter nor total egg volume once data had been adjusted for differences in fish size. These data suggest that despite very large differences in food availability throughout the periods of sexual differentiation and on-growing, investment in reproduction remained relatively consistent. It appeared that during food restriction, T. zillii sacrificed body weight and growth so as to maintain reproductive investment. In summary, this study provides valuable and novel information regarding the reproductive physiology and endocrinology of female T. zillii and suggests that this species may be a suitable 'model' species for future work on fecundity and ovarian development

Dissociation, enrichment, and the in vitro formation of gonocyte colonies from cryopreserved neonatal bovine testicular tissues

Gonocytes play an important role in early development of spermatogonial stem cells and fertility preservation to acquire more high quality gonocytes in vitro for further germ cell-related research and applications, it is necessarily needed to enrich and in vitro propagate gonocytes from cryopreserved bovine testicular tissues. This study aimed to investigate the isolation, enrichment, and colony formation of gonocytes in vitro for germ cell expansion from cryopreserved neonatal bovine testicular tissues. The effects of several different in vitro culture conditions, including seeding density, temperature, serum replacement and extracellular matrices were investigated for the maintenance, proliferation and formation of gonocyte colonies in vitro. Frozen/thawed two-week-old neonatal bovine testicular tissues were digested and gonocytes were enriched using a Percoll density gradient. Cell viability was accessed by trypan blue staining and cell apoptosis was evaluated by TUNEL assays. Gonocytes were identified and confirmed by immunofluorescence with the PGP9.5 germ cell marker and the OCT4 pluripotency marker while Sertoli cells were stained with vimentin. We found that neonatal bovine gonocytes were efficiently enriched by a 30%–40% Percoll density gradient (p < 0.05). No significant differences were detected between neonatal bovine testicular cells cultured at 34 °C or 37 °C. The formation of gonocyte colonies was observed in culture medium supplemented with knockout serum replacement (KSR), but not fetal bovine serum (FBS), at a seeding density higher than 5.0 × 104 cells/well. A greater number of gonocyte colonies were observed in culture plates coated with laminin (38.00 ± 6.24/well) and Matrigel (38.67 ± 3.78/well) when compared to plates coated with collagen IV and fibronectin (p < 0.05). In conclusion, bovine neonatal gonocytes were able to be efficiently isolated, enriched and maintained in gonocyte colonies in vitro; the development of this protocol provides vital information for the clinical translation of this technology and the future restoration of human fertility

Development of a laser-activated mesoporous silica nanocarrier delivery system for applications in molecular and genetic research

Nanoparticles have revolutionized medical research over the last decade. One notable emerging area of nanomedicine is research developments in the reproductive sciences. Since increasing evidence indicates links between abnormal gene expression and previously unexplained states of infertility, there is a strong impetus to develop novel tools, such as nanoparticle platforms, to elucidate the pathophysiological mechanisms underlying such states. Mesoporous silica nanoparticles (MSNPs) represent a powerful and safe delivery tool for molecular and genetic investigations. Nevertheless, on-going progress is restricted by low efficiency and unpredictable control of cargo delivery. Here, we describe for the first time, the development of a laser-activated MSNP system with heat-responsive cargo. Data derived from human embryonic kidney cells (HEK293T) cells indicate that when driven by a heat-shock promoter, MSNP cargo exhibits significantly increased expression following infra-red laser stimulus to stimulate a heat-shock response, without adverse cytotoxic effects. This new delivery platform with increased efficiency and the ability to impart spatial and temporal control is highly useful for molecular and genetic investigations. We envision that this new straightforward stimuli-responsive system could play a significant role in developing efficient nanodevices for research applications, for example in reproductive medicine

Determining the optimal time interval between sample acquisition and cryopreservation when processing immature testicular tissue to preserve fertility

The cryopreservation of immature testicular tissue (ITT) prior to gonadotoxic therapy is crucial for fertility preservation in prepubertal boys with cancer. However, the optimal holding time between tissue collection and cryopreservation has yet to be elucidated. Using the bovine model, we investigated four holding times (1, 6, 24, and 48 h) for ITTs before cryopreservation. Biopsies from two-week-old calves were stored in transport medium and cryopreserved following a standard slow-freezing clinical protocol. Thawed samples were then assessed for viability, morphology, and gene expression by haematoxylin and eosin (H&E) staining, immunohistochemistry and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Analysis failed to identify any significant changes in cell viability when compared between the different groups. Sertoli (Vimentin+) and proliferating cells (Ki67+) were well-preserved. The expression of genes related to germ cells, spermatogenesis (STRA8, PLZF, GFRα-1, C-KIT, THY1, UCHL-1, NANOG, OCT-4, CREM), and apoptosis (HSP70-2) remained stable over 48 h. However, seminiferous cord detachment increased significantly in the 48-h group (p < 0.05), with associated cord and SSC shrinkage. Collectively, our analyses indicate that bovine ITTs can be stored for up to 48 h prior to cryopreservation with no impact on cell viability and the expression levels of key genes. However, to preserve the morphology of frozen-thawed tissue, the ideal processing time would be within 24 h. Testicular tissues obtained from patients for fertility preservation often need to be transported over long distances to be cryopreserved in specialist centres. Our findings highlight the importance of determining optimal tissue transport times to ensure tissue quality in cryopreservation

Dynamic shapes of the zygote and two-cell mouse and human

Mouse zygote morphokinetics were measured during interphase, the mitotic period, cytokinesis, and two-cell stage. Sequences of rounder–distorted–rounder shapes were revealed, as were changing patterns of cross section area. A calcium chelator and an actin-disrupting agent inhibited the area changes that occurred between pronuclear envelope breakdown and cytokinesis. During cell division, two vortices developed in each nascent cell and they rotated in opposite directions at each end of the cell, a pattern that sometimes persisted for up to 10 h. Exchange with the environment may have been promoted by these shape and area cycles and persisting circulation in the cytoplasm may have a similar function between a cell's interior and periphery. Some of these movements were sporadically also seen in human zygotes with abnormal numbers of pronuclei and the two-cell stages that developed from these compromised human zygotes

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Search for gravitational waves associated with the InterPlanetary Network short gamma ray bursts

We outline the scientific motivation behind a search for gravitational waves associated with short gamma ray bursts detected by the InterPlanetary Network (IPN) during LIGO's fifth science run and Virgo's first science run. The IPN localisation of short gamma ray bursts is limited to extended error boxes of different shapes and sizes and a search on these error boxes poses a series of challenges for data analysis. We will discuss these challenges and outline the methods to optimise the search over these error boxes.Comment: Methods paper; Proceedings for Eduardo Amaldi 9 Conference on Gravitational Waves, July 2011, Cardiff, U

Protocol for developing a core outcome set for male infertility research:an international consensus development study

Abstract STUDY QUESTION We aim to develop, disseminate and implement a minimum data set, known as a core outcome set, for future male infertility research. WHAT IS KNOWN ALREADY Research into male infertility can be challenging to design, conduct and report. Evidence from randomized trials can be difficult to interpret and of limited ability to inform clinical practice for numerous reasons. These may include complex issues, such as variation in outcome measures and outcome reporting bias, as well as failure to consider the perspectives of men and their partners with lived experience of fertility problems. Previously, the Core Outcome Measure for Infertility Trials (COMMIT) initiative, an international consortium of researchers, healthcare professionals and people with fertility problems, has developed a core outcome set for general infertility research. Now, a bespoke core outcome set for male infertility is required to address the unique challenges pertinent to male infertility research. STUDY DESIGN, SIZE, DURATION Stakeholders, including healthcare professionals, allied healthcare professionals, scientists, researchers and people with fertility problems, will be invited to participate. Formal consensus science methods will be used, including the modified Delphi method, modified Nominal Group Technique and the National Institutes of Health’s consensus development conference. PARTICIPANTS/MATERIALS, SETTING, METHODS An international steering group, including the relevant stakeholders outlined above, has been established to guide the development of this core outcome set. Possible core outcomes will be identified by undertaking a systematic review of randomized controlled trials evaluating potential treatments for male factor infertility. These outcomes will be entered into a modified Delphi method. Repeated reflection and re-scoring should promote convergence towards consensus outcomes, which will be prioritized during a consensus development meeting to identify a final core outcome set. We will establish standardized definitions and recommend high-quality measurement instruments for individual core outcomes. STUDY FUNDING/COMPETING INTEREST(S) This work has been supported by the Urology Foundation small project award, 2021. C.L.R.B. is the recipient of a BMGF grant and received consultancy fees from Exscentia and Exceed sperm testing, paid to the University of Dundee and speaking fees or honoraria paid personally by Ferring, Copper Surgical and RBMO. S.B. received royalties from Cambridge University Press, Speaker honoraria for Obstetrical and Gynaecological Society of Singapore, Merk SMART Masterclass and Merk FERRING Forum, paid to the University of Aberdeen. Payment for leadership roles within NHS Grampian, previously paid to self, now paid to University of Aberdeen. An Honorarium is received as Editor in Chief of Human Reproduction Open. M.L.E. is an advisor to the companies Hannah and Ro. B.W.M. received an investigator grant from the NHMRC, No: GNT1176437 is a paid consultant for ObsEva and has received research funding from Ferring and Merck. R.R.H. received royalties from Elsevier for a book, consultancy fees from Glyciome, and presentation fees from GryNumber Health and Aytu Bioscience. Aytu Bioscience also funded MiOXYS systems and sensors. Attendance at Fertility 2020 and Roadshow South Africa by Ralf Henkel was funded by LogixX Pharma Ltd. R.R.H. is also Editor in Chief of Andrologia and has been an employee of LogixX Pharma Ltd. since 2020. M.S.K. is an associate editor with Human Reproduction Open. K.Mc.E. received an honoraria for lectures from Bayer and Pharmasure in 2019 and payment for an ESHRE grant review in 2019. His attendance at ESHRE 2019 and AUA 2019 was sponsored by Pharmasure and Bayer, respectively. The remaining authors declare no competing interests. TRIAL REGISTRATION NUMBER Core Outcome Measures in Effectiveness Trials (COMET) initiative registration No: 1586. Available at www.comet-initiative.org/Studies/Details/1586. TRIAL REGISTRATION DATE N/A. DATE OF FIRST PATIENT’S ENROLMENT N/A

A Micro RNA Processing Defect in Rapidly Progressing Idiopathic Pulmonary Fibrosis

BACKGROUND: Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF. METHODOLOGY AND PRINCIPAL FINDINGS: miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts. CONCLUSION: These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention